Various bioactive peptides have been reported in both the scientific literature and in issued patents. Peptides historically have been isolated from natural sources, and have recently been the subject of structure-function relationship studies. Additionally, natural peptides have served as starting points for the design of synthetic peptide analogs.
A review of peptide antibiotics was published by R. E. W. Hancock in 1997 (Lancet 349: 418-422). The structure, function, and clinical applications of various classes of peptides were discussed. An additional review of cationic peptide antibiotics was published in 1998 (Hancock, R. E. W. and Lehrer, R. Trends Biotechnol. 16: 82-88). The peptides are typically cationic amphipathic molecules of 12 to 45 amino acids in length. The peptides permeabilize cell membranes leading to the control of microbial agents. The clinical potential of host defense cationic peptides was discussed by R. E. W. Hancock in 1999 (Drugs 57(4): 469-473; Antimicrobial Agents and Chemotherapy 43(6): 1317-1323). The antibacterial, antifungal, antiviral, anticancer, and wound healing properties of the class of peptides are discussed.
Reviews of the structural features of helical antimicrobial peptides, and their presumed mechanisms of action have been published (see, for example, Dathe, M. and Wieprecht, T. Biochimica et Biophysica Acta 1462: 71-87 (1999); Epand, R. M. and Vogel H. J. Biochimica et Biophysica Acta 1462: 11-28 (1999)). Structural parameters believed to be capable of modulating activity and selectivity include helicity, hydrophobic moment, hydrophobicity, angle subtended by the hydrophilic/hydrophobic helix surfaces, and charge.
A wide array of naturally occurring alpha helical peptides have been reported. The following are representative of the many references in the field.
Cecropins are a family of α-helical peptides isolated from insects. Cecropins are known for their antibacterial properties, as described in U.S. Pat. Nos. 4,355,104 and 4,520,016. The cecropins were generally found to have activity against gram-negative bacteria, but not against all gram-negative bacteria. Cecropins were found not to have activity against eucaryotic cells (Andreu, et al., Biochemistry 24: 163-188 (1985); Boman, et al., Developmental and Comparative Immunol. 9: 551-558 (1985); Steiner et al., Nature 292: 246-248 (1981)). Cecropins from Drosophila and Hyalphora were presented as having activity against various strains of fungi (Ekengren, S. and Hultmark, D., Insect Biochem. and Molec. Biol. 29: 965-972 (1999)). Cecropin A from mosquito Aedes aegypti is reportedly different from most insect cecropins in that it lacks tryptophan and C-terminal amidation (Lowenberger, C. et al., J. Biol. Chem. 274(29): 20092-20097 (1999)).
Frogs from the genus Rana produce a wide array of antimicrobial peptides in their skin (Goraya, J. et al., Eur. J. Biochem. 267: 894-900 (2000)). Peptides as short as 13 amino acids were reported, and were grouped into structural families. The sequences showed little or no sequence identity to peptides isolated from frogs of other genera, such as the magainin and dermaseptin peptides.
U.S. Pat. No. 5,962,410 disclosed the inhibition of eucaryotic pathogens, and the stimulation of lymphocytes and fibroblasts with lytic peptides such as cecropins and sarcotoxins. Various peptides presented include Cecropin B, Cecropin SB-37, Cecropin A, Cecropin D, Shiva-1, Lepidopteran, Sarcotoxin 1A, Sarcotoxin 1B, and Sarcotoxin 1C.
Transgenic mice producing the Shiva-1 cecropin class lytic peptide were reported by Reed, W. A. et al., Transgenic Res. 6: 337-347 (1997). Infection of the transgenic mice with a Brucella abortus challenge resulted in a reduction of the number of bacteria relative to infection of non-transgenic mice.
Magainin is an α-helical 23 amino acid peptide isolated from the skin of the African frog Xenopus laevis (Zasloff, M. Proc. Natl. Acad. Sci. U.S.A. 84: 5449-5453 (1987).
Cathelin associated α-helical peptides of 23 to 38 amino acids are found in the blood cells of sheep, humans, cattle, pigs, mice, and rabbits (Zanetti, M. et al., FEBS Lett. 374: 1-5 (1995)).
The antimicrobial activities of buforin II, cecropin P1, indolicidin, magainin II, nisin, and ranalexin were reported by Giacomette, A. et al. (Peptides 20: 1265-1273 (1999)). The peptides showed variable activities against bacteria and yeast.
Various synthetic peptides have been prepared and assayed both in vitro and in vivo.
U.S. Pat. No. 5,861,478 disclosed synthetic lytic peptides of about 20 to 40 amino acids which adopt an α-helical conformation. The peptides are effective in the treatment of microbial infections, wounds, and cancer. The peptides disclosed include cecropin B, SB-37*, LSB-37, SB-37, Shiva 1 and 10-12, β-fibrin signal peptide, Manitou 1-2, Hecate 1-3, Anubis 1-5 and 8, and Vishnu 1-3 and 8.
Hecate was described as a synthetic peptide analog of melittin by Baghian, A. et al. (Peptides 18(2): 177-183 (1997)). The peptides differ in their charge distribution, but not in their amphipathic alpha helical conformation. Hecate inhibited herpes simplex virus (HSV-1) while not adversely affecting cell growth and protein synthesis.
Synthetic peptides D2A21, D4E1, D2A22, D5C, D5C1, D4E, and D4B were described in Schwab, U. et al., Antimicrob. Agents and Chemotherapy 43(6): 1435-1440 (1999). Activities against various bacterial strains were presented.
Hybrid peptides made of cecropin and melittin peptides were reportedly prepared and assayed by Juvvadi, P. et al. (J. Peptide Res. 53: 244-251 (1999)). Hybrids were synthesized to investigate the effects of sequence, amide bond direction (helix dipole), charge, amphipathicity, and hydrophobicity on channel forming ability and on antibacterial activity. Sequence and amide bond direction were suggested to be important structural requirements for the activity of the hybrids.
A 26 amino acid insect cecropin—bee melittin hybrid, and analogs thereof, were described in a study of salt resistance (Friedrich, C. et al., Antimicrobial Agents and Chemotherapy 43(7): 1542-1548 (1999)). A tryptophan residue in the second position was found to be critical for activity. Modest changes in sequence were found to lead to substantial changes in the properties of the peptides.
The effects of proline residues on the antibacterial properties of α-helical peptides has been published (Zhang, L. et al., Biochem. 38: 8102-8111 (1999)). The addition of prolines was reported to change the membrane insertion properties, and the replacement of a single proline may change an antimicrobial peptide into a toxin.
A series of peptides having between 18 and 30 amino acids were prepared in order to test the effects of changes in sequence and charge on antibacterial properties (Scott, M. G., et al., Infect. Immun. 67(4): 2005-2009 (1999)). No significant correlation was found between length, charge, or hydrophobicity and the antimicrobial activity of the peptides. A general trend was found that shorter peptides were less active than longer peptides, although the authors expressed that this effect would probably be sequence dependent.
“Modellins”, a group of synthetic peptides were prepared and assayed to compare sequence and structure relationships (Bessalle, R. et al. J. Med. Chem. 36: 1203-1209 (1993)). Peptides of 16 and 17 amino acids having hydrophobic and hydrophilic opposite faces were highly hemolytic and antibacterial. Smaller peptides tended to have lower biological activities.
A cecropin-melittin hybrid peptide and an amidated flounder peptide were found to protect salmon from Vibrio anguillarum infections in vivo (Jia, X. et al., Appl. Environ. Microbiol. 66(5): 1928-1932 (2000)). Osmotic pumps were used to deliver a continuous dose of either peptide to the fish.
Amphipathic peptides have been reported as being capable of enhancing wound healing and stimulating fibroblast and keratinocyte growth in vivo (U.S. Pat. Nos. 6,001,805 and 5,561,107). Transgenic plants have been reportedly prepared expressing lytic peptides as a fusion protein with ubiquitin (U.S. Pat. No. 6,084,156). Methylated lysine rich lytic peptides were reportedly prepared, displaying improved proteolytic resistance (U.S. Pat. No. 5,717,064).
While a number of natural and synthetic peptides exist, there exists a need for improved bioactive peptides and methods for their use.